A General Strategy for the Assignment of Aliphatic Side-Chain Resonances of Uniformly ¹³C,¹⁵N-Labeled Large Proteins

A general strategy is proposed to assign aliphatic side-chain resonances of large ¹³C,¹⁵N-labeled proteins without deuteration, using 4D ¹³C,¹⁵N-edited NOESY and MQ-(H)CCH-TOCSY experiments on the basis of prior assignments of backbone and ¹³C^β resonances. The strategy has been tested on a 214 residue protein (DdCAD-1) and applied to a chain-selectively ¹³C,¹⁵N-labeled hemoglobin (65 kDa). About 96 and 80% aliphatic side-chain spins in DdCAD-1 and hemoglobin have been assigned, respectively. The strategy proposed here will be very useful for the structure determination and dynamics characterization of large proteins by NMR.